Mass spectrometry (MS) has become an essential analytical tool due to its high sensitivity and ability to detect and identify a large number of analytes while also providing structural information of various molecules, which is very useful especially for biological sample analysis. Liquid chromatography (LC) is the advanced separation technique to resolve different species dissolved in the liquid phase, principally based on the difference of molecular properties.
LC-MS, a combination of powerful separation and identification techniques, is currently widely used in a broad range of fields, including pharma, biopharma, clinical, environmental, food safety, forensic, omics (proteomics, metabolomics, genomics, glycomics, and so on), academic research, regulatory agents, etc. As the front end tool, LC separates the components present in the sample and/or reduces the sample complexity before entering into MS for detection.
Electrospray ionization (ESI) is a technique to produce gas phase ions from liquid solution while applying a high voltage to create aerosol. ESI is commonly used as the interface to couple liquid phase separation methods (LC and CE) with MS. The ESI emitter, typically a needle or capillary made from fused silica, metal, or glass, connects with the LC column outlet and is located in the front of MS. As a soft ion source, ESI can create multiply charged molecule ions from LC elution, which provide the molecular weight information of the analytes of interest and are prone to conduct tandem MS (e.g., MS2) to explore their structure information.
ESI interfaces play an important role on LC-MS sensitivity. It is well known that ion loss during electrospray is one of the major causes affecting MS detection sensitivity. ESI efficiency or ionization efficiency increases with the decrease of the liquid flow rate and can approach 100% at low nL/min flow rates [Smith, R. D.; Shen, Y.; Tang, K. Acc. Chem. Res. 2004, 37, 269-278]. Very often, limited sample amounts, sample complexity, and the quest for highest possible MS sensitivity require the use of small inner diameter columns from 1.0-mm down to 20 μm, with corresponding operating flow rates from 100 μL/min to less than 20 nL/min. With a decrease in column inner diameter, the operating flow rate also decreases, which is more suitable for high sensitive MS detection.
An ESI interface also affects the LC separation performance because it introduces the post-separation extra-column dwell volume (dead volume) due to required connections, which will broaden the peaks and, therefore, decrease the resolution, signal intensity, and also impact MS detection (e.g. data-dependent MS2). Minimizing such extra column dwell volume is essential to achieve desired LC separation and MS results. Although small transfer lines are used to connect the column and emitter when using traditional ESI source, the presence of too many connections and transfer lines still affects the quality of data. This impact becomes more significant for low flow columns, particularly nano columns.
Typically the nano spray emitter is directly attached to the nano column outlet using a union to minimize the extra-column dwell volume (no extra transfer line and few connections). Currently, most end-users assemble the nano spray emitter with the nano LC column by themselves using the sleeves and fittings. However, this approach is prone to improper connections resulting in leaks, column/emitter breaks, or undesired dead-volumes, and consequently poor chromatographic separation and detection sensitivity. Also it may be difficult to achieve results with good reproducibility due to large variation of the emitter dimension and connections. The plug unit and connection system disclosed in US 2015/9091693 (e.g. nanoViper™ fitting, Thermo Fisher Scientific, Waltham, Mass., USA) has a virtually zero dead volume for connecting capillary tubes. It is convenient to achieve reliable connections, especially for ultra-high pressure liquid chromatography.
Besides selecting the correct LC column for desired separations, e.g. a nano LC column, minimizing, even eliminating, user intervention with the analytical techniques is also demanded to ensure reproducible results. A microchip based LC-ESI device integrates a trapping column, a separation column, and an electrospray emitter within a single structure [Fortier, M. H.; Bonneil, E.; Goodley, P.; Thibault, P. Anal. Chem. 2005, 77, 1631-1640]. While this integrated system has less dead volume, the chromatographic performance is currently not able to compete with that of conventional non-chip based systems due to the technology limitations and low column pressure rating.
The EASY-Spray™ column (Thermo Fisher Scientific, Waltham, Mass., USA) is an integrated system mainly containing a separation column and heating unit embedded in a plastic material, and an electrospray emitter which is connected with the separation column and protected by a retractable sleeve. [WO 2013/167131]. Because the connection of the column and emitter is embedded in the plastic material, the emitter is not able to be replaced when it fails, for example, due to clogging. In this case, the entire system is unusable even though the separation column is still functional.
To overcome the above challenges, it is desired to develop a stand-alone emitter assembly that provides a low dead-volume connection between the separation column and the mass spectrometer (or ion source of the mass spectrometer), is compatible with a broad range of flow rates, including nano LC-MS applications, and is easily replaced.